Screening, identification and characterisation of bacteriocins produced by the wine isolated LAB

Ndlovu, Joseph Buyani (2013-03)

Thesis (MSc)--Stellenbosch University, 2013.

Thesis

ENGLISH ABSTRACT: Lactic acid bacteria (LAB) play a vital role in reducing wine acidity and also contributing to its aroma and flavour. However, they can also be responsible for many wine spoilage problems that compromise the quality and value of wine. While Oenococcus oeni contributes positive characteristics to the sensory properties of wine, certain species of the genera, Lactobacillus and Pediococcus can affect the wholesomeness of wine by producing undesirable compounds, such as biogenic amines and ethyl carbamate. Chemical preservatives like sulphur dioxide (SO2) are used to prevent the growth of spoilage micro-organisms during the winemaking process. SO2 also acts as a reducing agent and maintains the benefits of antioxidant properties of the polyphenols of wine. However, there is a worldwide demand to reduce SO2 levels due to the increasing health related risks and other factors. All these considerations have increased the interest in research to look for new preservation strategies, and LAB-produced bacteriocins seem to be a potential alternative that has been explored in the last decade. Various types of bacteriocins have been identified and characterized. However, there are few reports on bacteriocins produced by LAB of oenological origin or on bacteriocins present in the finished wine. The present study screened 155 LAB isolates from the IWBT culture collection for bacteriocin production. The isolates originated from South African red wines undergoing spontenous malolactic fermentation (MLF). Eight strains (5%) were identified to be producers, as evidenced by strong inhibition zones formed against sensitive organisms on agar plates. The producers demonstrated a broad spectrum of antimicrobial activity by inhibiting Lactobacillus spp., Leuconostoc mesenteroides, Listeria monocytogenes and Pediococcus pentosaceus strains. Some of these bacterial genera are important in winemaking since they are potential wine spoilage bacteria. Hence these strains and/or the bacteriocins they produce could possibly find application in the food fermentation industry. The physiological results, biochemical tests and sugar fermentation profiles all gave the same results for the seven isolates, which were indicative of enterococci. The identification through 16S rRNA gene sequencing revealed that the seven tested isolates were all Enterococccus faecium. RAPD-PCR fingerprinting gave the same profile for the seven strains confirming that they were all identical on genetic level. Determining the molecular weight using SDS-PAGE showed the peptides to be below 4.6 kDa in size. PCR amplification of the enterocin P gene, sequencing and BLAST search results confirmed that all eight strains contained the enterocin P gene from Ent. faecium. The enterocin tested in this study was heat stable at 100°C (30 min), but lost 50% of its activity at 121°C (15 min). Factors such as bacteriocin production and heat resistance are among many that enable enterococci to be dominant in fermented products such as dairy foods or meat. Therefore, enterococci producing bacteriocins have potential applications in various foods and fermented products. The pH tests showed enterocin to be active over a broad pH range (2-10). Enterocin activity over a wide pH range make them potentially more suitable as natural preservatives of foods and fermented products where products are acidified or pH decreases due to natural LAB present. They also have potential applications in oenological process where pH levels are as low as 3 and 4. Proteolytic enzyme treatments with lysozyme, lipase, lyticase and catalase could not inhibit enterocin activity. This indicated that their antimicrobial activity was independent of lipid or carbohydrate moieties or hydrogen peroxide. α-Chymotrypsin and proteinase K inactivated enterocin, which indicated that the compound was proteinaceous in nature. Bacteriocin production tested in two of the isolates, #16.3 and 128.1, coincided with the exponential growth phase which occurred after 6 hours of incubation at 30°C, which was an indication of primary metabolite kinetics. The highest production of 400 AU/ml was observed after eight hours and was maintained for several hours (46 hours) in the stationery phase. The bactericidal effect of the cell free supernatants from #16.3 and 128.1 against the sensitive culture of Lactobacillus pentosus DSM 20314 was clearly demonstrated by complete inhibition of growth for most of the experimental period, while the control increased exponentially throughout the experiment. In conclusion, this study has confirmed the isolation and identification of Ent. faecium strains from wine, a genus that is rarely found in the wine environment. Although one can speculate on the origin of this bacterium in the wine e.g. human handling and contaminated water, these bacterial isolates produced enterocin P which have antimicrobial action against wine-related LAB genera and therefore have a potential role in wine spoilage control.

AFRIKAANSE OPSOMMING: Melksuurbakterieë (MSB) speel ‘n belangrike rol in die redusering van die suurgehalte van wyn en dra ook by tot die aroma en smaak daarvan. Hulle kan egter ook verantwoordelik wees vir vele wynbederfprobleme wat die gehalte en waarde van wyn negatief beïnvloed. Hoewel Oenococcus oeni positiewe karaktertrekke aan die sensoriese eienskappe van wyn verleen, kan sekere spesies van die genus, Lactobacillus en Pediococcus, die heilsaamheid van wyn beïnvloed deur ongewenste verbindings, soos biogeniese amienes en etielkarbamaat, te produseer. Chemiese preserveermiddels, soos swaweldioksied (SO₂), word gebruik om die groei van bederfmikro-organismes tydens die wynbereidingsproses te voorkom. SO₂ fungeer ook as ‘n reduseermiddel en onderhou die voordele van die antioksidant eienskappe van die poli-fenole van wyn. Daar is egter ‘n wêreldwye vraag na die redusering van SO₂-vlakke as gevolg van die toename in gesondheidsverwante risiko’s en ander faktore. Al hierdie oorwegings het belangstelling in die navorsing van nuwe preserveringstrategieë laat toeneem en MSB-geproduseerde bakteriosiene lyk na ‘n potensiële alternatief wat in die laaste dekade ondersoek word. Verskeie tipes bakteriosiene is geïdentifiseer en getipeer. Daar is egter nog weinig gerapporteer oor bakteriosiene wat deur MSB van wynkundige oorsprong geproduseer is of oor bakteriosiene wat in afgeronde wyn teenwoordig is. Die huidige studie het 155 MSB isolate van die Instituut vir Wynbiotegnologie se kultuurversameling vir bakteriosien-produsering gegradeer. Agt stamme (5%) is as produseerders geïdentifiseer, soos gestaaf is deur sterk inhibisiesones wat teen sensitiewe organismes op agarplate gevorm het. Die produseerders het ‘n breë spektrum van antimikrobiese aktiwiteit by inhiberende Lactobacillus spp., Leuconostoc mesenteroides, Listeria monocytogenes en Pediococcus pentosaceus stamme gedemonstreer. Sommige van hierdie bakteriese genera is belangrik in wynbereiding, omdat dit potensiële wynbederfbakterieë is. Hierdie isolate en/of die bakteriosiene wat dit produseer, kan dus moontlik toepassing in die voedselfermentasiebedryf vind. Die fisiologiese resultate, biochemiese toetse en suikerfermentasieprofiele het almal dieselfde resultate vir die sewe isolate, wat indikatief van enterococci was, gelewer. Die identifisering deur 16S rRNA-basispaaropeenvolging het onthul dat die sewe getoetste isolate almal Enterococccus faecium was. RAPD-PKR-vingerafdrukke het dieselfde profiel vir die sewe rasse gelewer, wat bevestig dat die rasse almal identies op genetiese vlak was. Deur die molekulêre gewig vas te stel deur middel van SDSPAGE, het dit getoon dat die peptiede kleiner as 4.6 kDa in grootte is. PKR-amplifikasie van die enterosien-P geen, die bepaling van basispaaropeenvolging en BLAST-soekresultate het bevestig dat al agt rasse die enterosien-Pgeen van Ent. faecium bevat. Die enterosien wat in hierdie studie getoets is, was hitte-stabiel teen 100°C (30 min), maar het 50% van sy aktiwiteit teen 121°C (15 min) verloor. Faktore soos bakteriosienproduksie en hittebestandheid, is van die vele faktore wat enterococci in staat stel om dominant in gefermenteerde produkte, soos suiwelprodukte of vleis te wees. Enterococci wat bakteriosiene produseer het dus potensiële toepassings in verskeie kossoorte en gefermenteerde produkte. Die pH-toetse het getoon dat enterosien-P oor ‘n breë pH spektrum (2-10) aktief was. Enterosienaktiwiteit oor ‘n wye pH spektrum maak dit potensieel meer geskik as natuurlike preserveermiddels vir kossoorte en gefermenteerde produkte waar produkte versuur word of die pH afneem as gevolg van natuurlike MSB wat teenwoordig is. Dit het ook potensiële toepassings in enologiese prosessering waar pH-vlakke so laag as 3 en 4 is. Proteolitiese ensiembehandelings met lisosiem, lipase, litikase en katalase kon nie enterosienaktiwiteit inhibeer nie. Daar is getoon dat hul antimikrobiese aktiwiteit onafhanklik was van lipiede, koolhidraatdele óf waterstofperoksied. α-Chymotripsien en proteïenase-K het enterosien onaktief gemaak, wat getoon het dat die samestelling proteïenagtig van nature is. Bakteriosienproduksie wat in twee van die stamme #16.3 en 128.1 getoets is, het ooreengestem met die eksponensiële groeifase wat na 6 ure van inkubasie teen 30°C plaasgevind het, en wat ‘n aanduiding is van primêre metabolitiese kinetika. Die hoogste produksie van 400 AU/ml is na agt ure waargeneem en is vir etlike ure (46 uur) in die stasionêre fase gehandhaaf. Die bakterie-dodende effek van die selvrye supernatant van #16.3 en 128.1 teenoor die sensitiewe kultuur van Lactobacillus pentosus DSM 20314 is duidelik gedemonstreer deur totale inhibisie van groei vir die grootste deel van die eksperimentele periode, terwyl die kontrole eksponensieel deur die hele eksperiment toegeneem het. Hierdie studie het dus die isolering en identifisering van Ent. faecium-stamme, ‘n genus wat baie selde gevind word in ‘n wynomgewing, vanuit wyn bevestig. Alhoewel daar gespekuleer kan word oor die oorsprong van hierdie bakterie in wyn bv. menslike hantering en besmette water, het hierdie rasse wel enterosien geproduseer en daarom die potensiaal om ‘n rol te speel in beheer teen verskeie bederf-MSB-genera.

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